Research in this proposal focuses on the four critical steps needed to make a red cell: the specific red cell differentiation signaling (through the erythropoietin receptor), the specific red cell transcription factor (GAGA- 1), the acquisition of the heme and iron needed to make the specific red cell protein, hemoglobin, and the red cell membrane. Project I tests the hypothesis that the three major signaling pathways activated by the erythropoietin receptor (EpoR)-Ras/MAP kinase, hypothesis that the three major signaling pathways activated by the erythropoietin receptor (EpoR)-Ras/MAP kinase, STAT5, and PI-3' kinase/Akt, function in different ways to prevent apoptosis of erythroid progenitors. Knock-in mice will be used to isolate and test individual pathways and to assess the redundancy of the cytoplasmic domains of TpoR, PrlR and EpoR in signaling. Other studies will explore the roles of transcription factor phosphorylation and ras activation in EpoR signaling. Project II investigates the function of GATA-1, the pivotal nuclear regulatory factor for erythroid gene expression and development. Targeted mutagenesis of GATA-1 will assess the importance of N-finger DNA-binding to erythropoiesis, the function of the N-terminal activation domain, and the role of serine phosphorylation. In addition, a newly discovered GATA-1 target gene, ABC-me, which encodes a putative mitochondrial heme transporter, will be studied by gene knockout in mice and mouse erythroleukemia cells, and by isolation of the zebrafish homologue. Project III evaluates a novel iron exporter called ferroportin 1 in the mouse and zebrafish. The function of ferroportin1 in mammal-ian iron metabolism will be tested by targeted gene disruption and by matings of ferroportin1 (-/-) and other iron deficiency overload mice. Genetic screens in zebrafish will identify factors necessary for ferro-portin1 expression adjacent to developing blood-island and will find genes that interact functionally with ferro-portin1. Project IV addresses three questions in red cell membrane biology. The function of the ankyrin-1 regulatory domain and the redundancy of questions in red cell membrane biology. The function of the ankyrin-1 regulatory domain and the redundancy of ankyrins-1 and -3 will be investigated using gene knock-in techniques. A new bIII spectrin, which is located in the Golgi and cytoplasmic vesicles, will be studied to identify the associated vesicles, characterize spectrin-vesicle interactions, and test the consequences of blocking such interactions or disrupting the bIII spectrin gene. In addition, following on test the consequences of blocking such interactions or disrupting the bIII spectrin gene. In addition, following on preliminary evidence that band 3 is involved in erythroid cytokinesis, interactions of band 3 with spindle components or other proteins will be sought using "pull-down" and two-hybrid assays, and genetic enhancer and suppressor screens in zebrafish. It is anticipated that these studies will provide information of broad interest and importance to our understanding of growth factor signaling, gene regulation, heme and iron transport and the biology of the red cell membrane skeleton.